Angelica Cazaly

Activation of Natural Killer Cells During Acute Infection With Hepatitis C Virus

BACKGROUND & AIMS Natural killer (NK) cells are essential early after infection, not only for viral containment but also for timely and efficient induction of adaptive responses. An inhibitory effect of hepatitis C virus (HCV)-E2 proteins on NK cells has been reported, but the features of NK cell responses in the acute phase of hepatitis C are still largely undefined. Therefore, the aim of this study was to characterize the function and phenotype of NK cells in the acute phase of infection and compare individuals with chronic and self-limited outcomes. METHODS Twenty-two individuals with acute HCV infection, 14 with chronic evolution, and 8 with self-limited infection, were studied using NK phenotypic and functional assays. RESULTS An increased expression of NKG2D on both CD56(bright) and CD56(dim) NK cells was detected in patients with acute HCV, irrespective of the outcome, as compared with healthy controls. Also, interferon gamma production and cytotoxicity by NK cells were higher in individuals with acute HCV infection than in healthy controls. Subset analysis showed increased interferon gamma production in both NK cell subsets carrying group 1 and group 2 HLA-C-specific killer cell immunoglobulin-like receptors. However, increased CD107a was noted only on NK cells expressing the group 1 HLA-C-specific killer cell immunoglobulin-like receptor and was maximal in self-limited infection. CONCLUSIONS Our data show that in the acute phase of HCV infection, NK cells are activated regardless of outcome, with no evidence of a suppressive effect of HCV on NK cell function.

Peptide antagonism as a mechanism for NK cell activation

Inhibition of natural killer (NK) cells is mediated by MHC class I receptors including the killer cell Ig-like receptor (KIR). We demonstrate that HLA-C binding peptides can function as altered peptide ligands for KIR and antagonize the inhibition mediated by KIR2DL2/KIR2DL3. Antagonistic peptides promote clustering of KIR at the interface of effector and target cells, but do not result in inhibition of NK cells. Our data show that, as for T cells, small changes in the peptide content of MHC class I can regulate NK cell activity.

Colonic expression of leukotriene-pathway enzymes in inflammatory bowel diseases

Background Leukotrienes derived from the 5‐lipoxygenase pathway are proinflammatory lipid mediators that possibly play a role in inflammatory bowel diseases. The expression of 5‐lipoxygenase pathway proteins has not previously been examined in colonic mucosa in inflammatory bowel disease. Results Quantitative immunohistochemical analyses showed that, compared to those of the control subjects (n = 9), colonic biopsies from patients with active inflammatory bowel disease (n = 17) had 3‐ to 7‐fold higher mean counts of cells expressing 5‐lipoxygenase (P = 0.03), 5‐lipoxygenase‐activating protein (P = 0.005), and the leukotriene A4 hydrolase (P = 0.004), which make up the biosynthetic pathway of the potent neutrophil chemotaxin leukotriene B4. Immunoexpression of the leukotriene C4 synthase was unaltered (P > 0.2). The increased representation of leukotriene B4–pathway enzymes was associated with higher counts of neutrophils (P = 0.0001), macrophages (P = 0.03), eosinophils (P = 0.0004), CD8+ T cells (P < 0.001), activated T cells (P < 0.05), and B cells (P < 0.05) but not of mast cells (P > 0.9). These eicosanoid and cellular changes were most marked in the subgroup of patients with ulcerative colitis (n = 9), and were absent in patients with quiescent disease (n = 6). The anomalies in the 5‐lipoxygenase pathway were accompanied as expected by more cells immunostaining for cytokine‐inducible COX‐2 (P = 0.004, n = 17), but this study also revealed a greater number of cells expressing COX‐1 in the samples from the patients in the ulcerative colitis subgroup (P = 0.03, n = 9). Conclusions The 5‐lipoxygenase data provide a cellular basis for increased tissue synthesis of the leukotriene B4, as reflected in the colonic mucosa and rectal dialysates of patients with active inflammatory bowel disease, which contributes to neutrophil influx and colonic injury. The COX‐1/COX‐2 data highlight the ambiguous functional role of prostanoid pathways in inflammatory bowel diseases. (Inflamm Bowel Dis 2007)

Human bronchial epithelial cells express an active and inducible biosynthetic pathway for leukotrienes B4 and C4

Background Human bronchial epithelial cells synthesize cyclooxygenase and 15‐lipoxygenase products, but the 5‐lipoxygenase (5‐LO) pathway that generates the leukotriene (LT) family of bronchoconstrictor and pro‐inflammatory mediators is thought to be restricted to leucocytes.

Human bronchial fibroblasts express the 5-lipoxygenase pathway

BackgroundFibroblasts are implicated in sub-epithelial fibrosis in remodeled asthmatic airways and contribute to airway inflammation by releasing cytokines and other mediators. Fibroblast activity is influenced by members of the leukotriene family of bronchoconstrictor and inflammatory mediators, but it is not known whether human bronchial fibroblasts can synthesize leukotrienes.MethodsThe expression of leukotriene biosynthetic enzymes and receptors was investigated in primary fibroblasts from the bronchi of normal and asthmatic adult subjects using RT-PCR, Western blotting, immunocytochemistry and flow cytometry.ResultsThese techniques revealed that human bronchial fibroblasts from both subject groups constitutively express 5-lipoxygenase, its activating protein FLAP, the terminal enzymes leukotriene A4 hydrolase and leukotriene C4 synthase, and receptors for leukotriene B4 (BLT1) and cysteinyl-leukotrienes (CysLT1). Human bronchial fibroblasts generated immunoreactive leukotriene B4 and cysteinyl-leukotrienes spontaneously and in increased amounts after calcium-dependent activation. Flow cytometry showed that human bronchial fibroblasts transformed to a myofibroblast-like phenotype by culture with transforming growth factor-β1 expressed 320–400% more immunofluorescence for leukotriene C4 synthase and CysLT1 receptors, with 60–80% reductions in leukotriene A4 hydrolase and BLT1 receptors.ConclusionThese results indicate that human bronchial fibroblasts may not only respond to exogenous leukotrienes but also generate leukotrienes implicated in narrowing, inflammation and remodeling of the asthmatic airway.

Thinking Outside the Gate: Single-Cell Assessments in Multiple Dimensions

Present address: Immuno-Oncology & Combinations DPU, Oncology RDBendall et al., 2011), allowing an oppor-tunity to better understand the immuno-logical mechanisms underlying disease.Complex flow cytometry (FCM) data arenow surpassing our ability to fully analyzeand interpret all information via currentstandard approaches, such as 2D dotplots and Boolean gates. Indeed, thenumber of potential cell subpopulationsincreases exponentially with the numberof parameters assessed, making it diffi-cult to decipher all possible combina-tions included in the raw data (e.g., 512potential subsets with nine markers) viathe traditional approaches (Bendall andNolan, 2012). This could limit the transla-tion of technical advances into new diag-nostics or therapies. Newly developedbioinformatics tools that have the poten-tial to bridge this gap are now available.The aim of this letter is to foster theimplementation and adoption of thesenovel computational methodologies forunbiased analysis of complex cytometrydata.In recent years, a host of new data-analysis tools have emerged, creatingworkflows for processing and analyzingcomplex FCM datasets; however, thesehave gone mostly unnoticed by immunol-ogists. Table S1 provides an overview ofmany of the currently available tools andtheir specific applications. They can beassigned to specific categories arrangedin a ‘‘FCM data-analysis workflow’’ fromcompensated data as input to biologi-cally interpretable results as output. Thevast majority of the listed tools for FCMdata processing, analysis, and visualiza-tion are made available by the bio-informaticians at no cost and includeopen source code and unrestrictive soft-ware licensing, opening up these compu-tational approaches to broad use by theresearch community. Many of the toolshave been developed to address similaranalysis objectives via quite different ap-proaches. They might provide optimal re-sultsfordifferentdatasets,suchthatthereis no ‘‘right’’ or ‘‘best’’ tool, and usingseveral algorithms in combination mightyield even better results and exceedthe possibilities offered by manual anal-ysis.Comprehensivecomparativestudiesby the Flow Cytometry: Critical Assess-mentofPopulationIdentificationMethods(FlowCAP) project have shown that manyof these tools have reached a level ofmaturitythatmatches,orevensurpasses,the results produced by human experts(Aghaeepour et al., 2013).The development of computational ap-proaches addresses many needs asso-ciated with high-dimensional datasets.However,fortheimmunologycommunity,threemain challengeshave surfaced, andtackling them will facilitate a paradigmshift in the analysis of FCM data. First,despite the focused efforts by bio-informaticians to develop novel tools foranalyzingFCMdata,onlyaminorityofim-munologists are aware of the advantagesofferedtothefield.Thesetoolsneedtobepresented in immunology forums ratherthan limited to bioinformatics journalsand conferences. Second, even thoughthe vast majority of the computationalImmunity 42, April 21, 2015 a2015 Elsevier Inc. 591

Targeting Carcinoembryonic Antigen with DNA Vaccination: On-Target Adverse Events Link with Immunologic and Clinical Outcomes

Purpose: We have clinically evaluated a DNA fusion vaccine to target the HLA-A*0201–binding peptide CAP-1 from carcinoembryonic antigen (CEA605–613) linked to an immunostimulatory domain (DOM) from fragment C of tetanus toxin. Experimental Design: Twenty-seven patients with CEA-expressing carcinomas were recruited: 15 patients with measurable disease (arm-I) and 12 patients without radiological evidence of disease (arm-II). Six intramuscular vaccinations of naked DNA (1 mg/dose) were administered up to week 12. Clinical and immunologic follow-up was up to week 64 or clinical/radiological disease. Results: DOM-specific immune responses demonstrated successful vaccine delivery. All patients without measurable disease compared with 60% with advanced disease responded immunologically, while 58% and 20% expanded anti-CAP-1 CD8+ T cells, respectively. CAP-1–specific T cells were only detectable in the blood postvaccination but could also be identified in previously resected cancer tissue. The gastrointestinal adverse event diarrhea was reported by 48% of patients and linked to more frequent decreases in CEA (P < 0.001) and improved global immunologic responses [anti-DOM responses of greater magnitude (P < 0.001), frequency (P = 0.004), and duration] compared with patients without diarrhea. In advanced disease patients, decreases in CEA were associated with better overall survival (HR = 0.14, P = 0.017). CAP-1 peptide was detectable on MHC class I of normal bowel mucosa and primary colorectal cancer tissue by mass spectrometry, offering a mechanistic explanation for diarrhea through CD8+ T-cell attack. Conclusions: Our data suggest that DNA vaccination is able to overcome peripheral tolerance in normal and tumor tissue and warrants testing in combination studies, for example, by vaccinating in parallel to treatment with an anti-PD1 antibody. Clin Cancer Res; 22(19); 4827–36. ©2016 AACR.

Validation of immunomonitoring methods for application in clinical studies: The HLA-peptide multimer staining assay

Validated assays are essential to generate data with defined specificity, consistency, and reliability. Although the process of validation is required for applying immunoassays in the context of clinical studies, reports on systematic validation of in vitro T cell assays are scarce so far. We recently validated our HLA‐peptide multimer staining assay in a systematic manner so as to qualify the method for monitoring antigen‐specific T cell responses after immunotherapy.

Bone marrow transplantation for MHC class I deficiency corrects T-cell immunity but dissociates natural killer cell repertoire formation from function

The index datewas defined as the date of the first indicator of diabetes for cases and the date of selection for controls. The dose of ICSs was categorized as low (<251 mg/d, fluticasone propionate equivalents), medium (251-500 mg/d), high (>500 mg/d), or no use, and LABAwas classified as use (>_1 mg/d, salmeterol equivalents) or no use and measured on the index date. This study was approved by the Research and Ethics Committee of the Hôpital du Sacr e-Cœur de Montr eal and by the Commission d’acc es a l’information du Qu ebec. For additional methodological details, see this article’s Online Repository at www.jacionline.org. We identified 1001 cases of GD and selected 30,030 controls without GD among the 12,587 pregnancies of women with asthma (see Fig E1 in this article’s Online Repository at www. jacionline.org). The cases were older and more likely to have uncontrolled asthma and a history of GD (see Table E3 in this article’s Online Repository at www.jacionline.org). The generalized estimation equations models (Table I) indicate that the dose of ICSs among nonusers of LABAs was not significantly associated with GD. Moreover, adding LABAs to ICSs did not increase the risk of GD compared with ICSs alone. We also found that women who used a medium ICS dose without a LABAwere not significantly at a higher risk of GD than women who used a low ICS dose plus a LABA. Moreover, the risk of GD was found similar between women who used a high ICS dose without a LABA and those who used a medium ICS dose plus a LABA. Strengths of this study include a large sample size, avoiding recall bias, and fixing cohort entry at week 20 of gestation to avoid index event bias. Fixing cohort entry at pregnancy onset could have biased the odds ratios toward the null in case ICSs or LABAs would be associated with early pregnancy termination. Nevertheless, unmeasured covariates such as ethnicity, obesity, and familial history of GD may cause residual confounding if they are associated with the use of ICSs or LABAs. The indications for antiasthmatic medications may also introduce residual confounding because higher ICS doses or the addition of LABAs is likely to be prescribed to those with more severe asthma. However, despite this potential bias, the adjusted odds ratios associated with medium and high ICS doses and with LABAs remained close to the null and not statistically significant. Our results provide further evidence on the safety of LABAs and ICSs during pregnancy with no significantly increased risk of GD detected among women exposed to those medications. Although not statistically significant, the adjusted odds ratios associated with moderate and high doses of ICS were slightly greater than 1, and it might be due to residual confounding by obesity or asthma severity. More studies with better adjustment capacity for obesity and asthma severity are needed to further examine the association between higher ICS doses and the risk of GD in women with asthma.

Defects in lymphocyte subsets and serological memory persist a median of 10 years after high-dose therapy and autologous progenitor cell rescue for malignant lymphoma.

The number of survivors having undergone high-dose therapy (HDT) followed by auto-SCT continues to increase, although some of the long-term sequelae remain incompletely understood. The immunological status and quality of life of 37 HDT/auto-SCT survivors with lymphoma in continuous remission of ⩾3 years were assessed alongside 14 age-matched controls. At a median follow-up of 10.5 years (range 2.2–20.2) following HDT/auto-SCT, the proportion of CD4+ cells remained significantly reduced in patients compared with controls (median 43.4% vs 62.5%, respectively; P=<0.001), predominantly a result of sustained reduction in the naive CD4+ component (P<0.001). Naive CD8+ lymphocytes (P=0.014) and transitional B cells (P=0.008) were also significantly reduced, but differences in other lymphocyte subsets were not observed. Uptake of revaccination following HDT/auto-SCT was sporadic; between 11% and 33% of patients had serological titres outside the protective ranges for five of six routinely used vaccines. In the main, patients were found to have a good quality of life, although their EORTC QLQ-C30 questionnaire scores were significantly lower for the physical and social functioning domains compared with controls. Ten years after HDT/auto-SCT immunological deficits persist; to avoid excess risk of preventable disease, serological immunity should be assessed post HDT/auto-SCT followed by appropriate revaccination.